Gram-negative bacteria are prominent members of the microbial flora of all animals. When they invade into tissue, often through a break in the epithelium, they are quickly countered by the body's innate host defenses. Much of this beneficial response is triggered by a bacterial cell wall lipopolysaccharide (LPS) that, because it can also induce lethal reactions, has earned the name "endotoxin." The long-term goal of our research has been to learn how animals inactivate (detoxify) LPS. During the next funding period, we hope to define the biological function(s) of an animal enzyme, acyloxyacyl hydrolase (AOAH), that selectively cleaves the secondary fatty acyl chains from the lipid A region of LPS. Although enzymatically-deacylated LPS is inactive in several assay systems, suggesting that AOAH should detoxify LPS in vivo, the precise contribution of the enzyme to LPS detoxification is uncertain and the enzyme may have functions unrelated to its ability to deacylate LPS. The proposed research builds on 3 recent developments in our lab: the discovery that AOAH is present in immature dendritic cells and can deacylate E. coli that the cells ingest, the finding that AOAH is abundantly produced in the proximal tubule cells of the kidney and secreted into urine, and the targeted disruption of the AOAH gene to produce AOAH "knockout" mice. Our Specific Aims are (1) to determine the role of LPS deacylation in LPS inactivation, in vitro and in vivo, (2) to find out the function(s) of acyloxyacyl hydrolase (AOAH) in the kidney, and (3) to determine the role of LPS deacylation in the presentation of LPS and bacterial outer membrane proteins to B and T cells. Finding out the functions of this highly conserved enzyme should shed new light on how animals control their inflammatory and immune reactions to gram-negative bacteria.